DE69806962T2 - FSNMU as an anti-inflammatory agent in the surface tissues of mammals - Google Patents

Abstract

A topical pharmaceutical or cosmetic composition comprising, as the active ingredient, at least one of the NMIFA's having the following formula, wherein the NMIFA is under the form of an acid, a salt or an ester, and R1 is C1-C5 alkyl and R2 is C2-C6 alkyl, preferably wherein the NMIFA is 20:3(5,11,14). These NMIFA's are used for the preparation of a composition intended to modulate the metabolism of lipids in superficial mammal tissues. <CHEM>

Description

The present invention relates to the novel use of fatty acids that are not interrupted by methylene groups in the prevention and/or treatment of inflammation in the superficial tissues of mammals.

Background of the invention

The use of fatty acids to prevent or treat inflammation in superficial tissues has been described in the literature.

For example, EP 5 472 705 (Prospa B. V.) describes new pharmaceutical compositions for topical application containing esters of polyunsaturated ω-3 acids in high concentration and intended for the treatment of psoriasis, phlebitis and related diseases.

BP 582 239 (Rhöne Poulenc Rorer) describes pharmaceutical and cosmetic topical compositions containing linoleic acid or its derivatives as active ingredients and a vehicle for transporting the active ingredient into the skin. The compositions are used for the prevention and treatment of impure skin, for example skin affected by pimples, pustules, hives or blackheads, acne and skin diseases associated with acne.

Furthermore, EP 5 312 834 (Wooband Land Co.) describes a pharmaceutical composition for the treatment of acne, which contains eicosapentaenoic acid and α-linolenic acid in a weight ratio of eicosapentaenoic acid to α-linolenic acid of 1 : 0.1 to 20 : 0.1. This Fatty acids can be extracted from natural substances such as fish oil and/or perilla oil.

The term fatty acids not interrupted by methylene groups, whose acronym is FSNMU, refers to fatty acids containing a series of double bonds, with at least one adjacent pair of double bonds separated by at least two carbon atoms, namely by a group other than a single methylene group. FSNMU have been the subject of several studies solely to develop an understanding of their uptake into mammalian tissues and their potential role in the treatment of certain diseases.

For example, JP 61 058 536 (Nippon Oil) describes a process for purifying pine nut oil which contains at least 10% by weight of cis-5,9,12-octadecatrienoic acid and has a healing effect against arterial hypertension.

WO 96 05 164 (Broadbon Nominees Pty) describes an anti-inflammatory preparation comprising a purified active ingredient fraction, for example 5,11,14,17-eicosatetraenoic acid, isolated from a lipid extract of Perna canaliculus or of Mytilus edulis.

WO 95 17 897 (The Regents of the University of California) shows that the large class of FSNMU including 5,11,14-eicosatrienoic acid can be used in an amount effective to suppress autoimmune diseases in general, such as rheumatoid arthritis, lupus erythematosus, multiple sclerosis, myasthenia gravis and about 30 other currently known diseases.

The uptake of dietary 5,11,14-eicosatrienoate into various classes of phospholipids and tissues of mice was investigated. The results show that their supply with 5,11,14-eicosatrienoate resulted in a lower content of 20:4n-6 in the phosphatidylinositol depot of the liver. Considering that leukotrienes and prostaglandins cannot be formed from 5,11,14-eicosatrienoate due to the absence of an internal double bond Δ8 and considering that the content of 20:4n-6 is dramatically reduced in some phosphatidylinositol depots, it was expected that dietary intake of 5,11,14-eicosatrienoate may alter eicosanoid metabolism, thus reducing the possibility of inflammation in the liver system (Biochemica et Biophysica Acta, 1085, 371-376, 1991; J. Nutr. Biochem., 4, 409-420, 1993).

To date, it has not been described that the following class of FSNMU, and in particular 5,11,14-eicosatrienoic acid, can be incorporated into the lipid fraction of superficial tissues of mammals. Nor was an anti-inflammatory effect in the superficial tissues of mammals foreseeable.

Summary of the invention

It has been found that at least one of the FSNMUs of the following formula, wherein the FSNMU is an acid, salt or ester and wherein R¹ is a C₁₋₅ alkyl group and R² is a C₂₋₆ alkyl group, can be used to prepare a composition intended for modulating lipid metabolism in the superficial tissues of mammals:

Particularly preferred FSNMU are those compounds wherein R¹ is a C₃-alkyl group and R² is a C₂₋₆-alkyl group or wherein R² a C4 alkyl group and R1 a C1-5 alkyl group. Most preferred is the FSNMU wherein R1 is an n-propyl group and R2 is an n-butyl group (5,11,14-eicosatrienoic acid, also referred to as 20:3(5,11,14)).

In this context, the FSNMU can also be used to prepare a composition intended to treat or prevent inflammation in the superficial tissues of mammals by modulating lipid metabolism.

The invention further relates to pharmaceutical and cosmetic topical compositions containing the FSNMU of the invention as an active ingredient.

A pharmaceutical, food or cosmetic composition containing a combination of fish oil and FSNMU.

The FSNMUs offer similar advantages to those of fish oils known to the person skilled in the art. However, given that they have only two bonds interrupted by a methylene group compared to the docosahexaenoic acid contained in fish oil, which has 6 bonds interrupted by a methylene group, they offer the advantage of being less easily oxidized than fish oil. In addition, the FSNMUs of the invention are not a substrate for the formation of prostaglandin and leukotriene, whereas prostaglandin and leukotriene can be formed from the fatty acids present in fish oil, such as 20:5n-3 and 22:6n-3. Another advantage compared to the processing of a fish oil is that there is no "fishy smell".

Detailed description of the invention

"Modulation of lipid metabolism" refers in particular to the catabolism of lipid mediators that are involved in inflammation, differentiation, the proliferation and/or barrier function of the superficial tissues.

Inflammation of superficial tissues should also be understood as the physiological phenomenon involving the production of proinflammatory cytokines, such as cachectin α (TNF-α), by the cells of superficial tissues, for example the keratinocytes and the epithelial cells of the cornea, and the cells of the immune system (lymphocytes, Langerhans cells and the like) contained in these tissues. The inflammation can result, for example, from an infection, an allergy, an injury and the action of radiation and/or irritants and/or sensitizing agents.

The fatty acid usable in the present invention is a polyunsaturated fatty acid which is straight-chain and has a carboxyl group, with all double bonds being cis-double bonds. In the present description, several types of terminology are used, which are listed below:

(a) The terminology for each compound, indicating the number of carbon atoms and the number and position of double bonds; a typical example is "20 : 4(5,8,11,14)" for arachidonic acid. The number before the colon indicates the total number of carbon atoms, the number immediately after the colon indicates the number of double bonds and the numbers in brackets indicate the positions of the double bonds, starting from the end of the chain carrying the carboxylic acid group. Unless otherwise stated, in all compounds so designated, all double bonds are cis double bonds.

b) The terminology for the compound classes, which indicates the position of the double bond closest to the terminal methyl group; a typical example is "n-3" or "n-6". The number after the hyphen indicates the position of the double bond closest to the methyl end of the molecule, starting from the methyl end. Thus, arachidonic acid belongs to the class n-6, as does 5,11, 14-eicosatrienoic acid (20 : 3(5,11,14)), while 5,8,11, 14,17-eicosapentaenoic acid (20 : 5(5,8,11,14,17)) belongs to the class n-3. The terminology corresponds to the "w" terminology in the literature, where "ω" and "n" are interchangeable.

Some of the FSNMUs of the invention are substances that occur in nature. Others can be prepared by well-known published methodology (see, for example, Evans et al., Chem. Phys. Lipids, 38 327-342, 1995).

For example, 20:3(5,11,14) is a substance that occurs in nature, usually as a fatty acid in a mixture of fatty acids. This FSNMU is found in all plant species as a small or large fraction of the total composition of fatty acids. Both the extraction of the fatty acid mixture from the natural sources and the extraction of 20:3(5,11,14) from the resulting fatty acids can be accomplished using conventional extraction and purification procedures that are well known to those skilled in the art.

Natural sources of fatty acids containing 20:3(5,11,14) are mainly plant seeds, including in particular seeds of conifers and ornamental shrubs. The seed oils of these plants resemble normal cooking oils, containing mainly oleic acid, linoleic acid and linolenic acid, but also contain worthwhile amounts of FSNMU.

Table 1 lists examples of seeds whose lipid content includes significant amounts of 20:3(5,11,14). Table 1

*see Japanese patent JP 5276964 (Suntory Ltd.)

The purification of 20:3(5,11,14) can be carried out in particular by (1) initially selecting a seed source with a high total fat content and a high content of 20:3(5,11,14) but which does not contain other contaminating trienes and in particular α-linolenic acid (18:3n-3) and γ-linolenic acid (18:3n-6) (Podocarpus nagi, Table 1, is an example); (2) extracting the lipids using isopropanol and chloroform according to the method of Nichols (Biochim. Biophys. Acta 70:417, 1963); (3) using conventional decoction and decolorization procedures; (4) methyl esters can be prepared with a 2% sulphuric acid-methanol solution according to the method of Christie (pages 52-53, in Lipid Analysis, Pergamon Press, Oxford, 1982); (5) the methyl ester of 20:3(5,11,14) with a hexane/ether Mixture of 9:1 to 8:2 (volume/volume) is eluted on a Florisil column impregnated with silver nitrate and washed with acid (according to Carroll, J. Am. Oil Chem. Soc. 40: 413, 1963; Wilner, Chem. Ind. (Lond) October, 30 : 1839, 1965; Merck ChromNews 4(I), 1995; Anderson, J. Lipid Res. 6: 577, 1965 and Teshima, Bull. Jap. Soc. Scien. Fish. 44: 927, 1978); (6) the contaminating silver ions are removed by the method of Äkesson (Eur. J. Biochem. 9: 463, 1969) and (7) the methyl ester is optionally converted into potassium hydroxide 1M in ethanol (95%) according to Christie (pages 51-52, in Lipid Analysis, Pergamon Press, Oxford, 1982), whereby the free acid is formed by saponification.

In the context of the present invention, the FSNMU can be used in the form of an acid, a salt or an ester, for example as a methyl ester, ethyl ester, mono-, di- or triglyceride or phospholipid ester. The FSNMU of the invention can be used in purified form or also in the form of a mixture of fatty acids, which is present, for example, in an oil extracted from one of the plants described above.

The FSNMU can be administered by methods known to the person skilled in the art to all types of superficial tissues of mammals, in other words to cells which, for example, make up the skin, scalp, eye or the oral, tongue, nasal and vaginal mucosa.

The FSNMU can be used for the treatment or prophylaxis of diseases of the skin or scalp and in particular against inflammations associated with, for example, psoriasis, erythema (sunburn), eczema, seborrhoeic dermatitis, pelade, mycosis, acne and other skin diseases.

A composition intended for topical application may therefore comprise an effective amount of FSNMU as the active ingredient and a vehicle to deliver the active ingredient to the superficial tissues of mammals.

The vehicle can be any vehicle for cosmetic or pharmaceutical formulations known to the person skilled in the art. One of the vehicles used in this context can be liposomes. These molecules include one or more different types of substances, in particular non-polar lipids, polar lipids, mono- and diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids and salts. The liposomes can be in the form of emulsions and foams, micelles, insoluble monolayers, liquid crystals, dispersions of phospholipids and lamellar layers. The FSNMU can optionally be included in the liposome together with a ligand or a suitable mimetic that binds to the specific receptors of the cell.

The liposomes are usually composed of lipids that form standard vesicles, which generally include neutral phospholipids or phospholipids with a negative charge and a sterol such as cholesterol. The choice of lipids is usually made taking into account various factors, such as the desired size of the liposomes and the need for stability of the liposomes in the bloodstream. The main lipid component of the liposomes is typically phosphatidylcholine. A typical example is partially hydrogenated phosphatidylcholine from eggs.

The liposomes typically contain about 5 to 15 mol% of phospholipids with a negative charge, such as phosphatidylglycerol, phosphatidylserine or phosphatidylinositol. The phospholipids with a negative charge carry help to prevent spontaneous aggregation of the liposomes and thus reduce the presence of aggregates of liposomes that are undersized. Membrane stiffening agents such as sphingomyelin or a saturated neutral phospholipid may also be incorporated at a concentration of at least about 50 mol% and 5 to 15 mol% monosialylganglioside to achieve further distribution of the liposome preparation in the bloodstream.

The liposome suspensions may further contain lipid protecting agents to protect the lipids against damage by radicals and peroxides during storage. Examples of these agents are lipophilic free radical scavengers such as α-tocopherol and specific water-soluble iron chelating agents such as ferrioxamine.

The liposomes can be produced using any method known to the person skilled in the art. The liposomes can then be dimensioned within a desired particle size range and with a relatively narrow particle size distribution. A particle size range that allows the liposome suspension to be sterilized by filtration through a conventional filter is, for example, the range from about 0.2 to 0.4 micrometers (μm).

The formulations intended for administration generally contain a solution of the compound in solution or suspension in an acceptable vehicle, the appropriate choice being easily made by the person skilled in the art. The formulations may also contain pharmaceutically acceptable auxiliary substances which may be required to approximate the physiological conditions, such as pH adjusters and buffers, tonicity adjusters and wetting agents. The formulations may also advantageously contain antioxidants and preferably fat-soluble Antioxidants, such as in particular tocopherol, tocopheryl acetate, butylhydroxytoluene, butylhydroxyanisole and ascorbyl palmitate and mixtures of these compounds.

The cosmetic or pharmaceutical composition can be applied directly to the superficial tissues, allowing the FSNMU to penetrate through the cells and be released there. The composition can also be injected beneath the superficial tissues, for example by subcutaneous injection. In both cases, the application is considered topical, in other words, the application is made directly on or beneath the superficial tissues.

The application of FSNMU can also be extended to inflammation of the eye and in particular the cornea, as well as the mucous membranes and in particular the mucous membranes of the mouth, nose, tongue, anus and vagina. The orally administered compositions can act directly on the mucous membranes of the tongue, the mucous membranes of the esophagus, the mucous membranes of the stomach and the intestinal mucosa and/or they can enter the bloodstream and be transported directly to the cells of the skin, eye or mucous membrane, for example.

The composition can also be applied into the nasal passages using a nebulizer, a gel and/or a physiological liquid and used in the conventional way to rinse the nasal passages.

It should be stressed that the amount of FSNMU necessary and sufficient to obtain an anti-inflammatory effect or a modulatory effect on lipid metabolism varies depending on the galenic forms commonly used for topical or oral application, in other words, depending on whether it is the form of a food, a cosmetic and/or a pharmaceutical form, can vary considerably. The use of FSNMU in an amount sufficient for the treatment or prevention of inflammation and/or for the modulation of the lipid metabolism of the superficial tissues.

The FSNMU are administered to humans or animals for the treatment of the oral mucosa, for example in the form of a food. They can be used as a substitute for the oils commonly used in food preparations or recipes, or as part of a mixture of oils used in this way. The composition can be, for example, a sauce such as a salad dressing, a table oil, a mayonnaise, an ice cream, a confectionery composition or a paste for garnishing or spreading. The compound can also be administered as part of a nutritional supplement such as a tablet or a gelatin capsule taken orally on a daily basis. This usually also includes binders, matrices and other conventional additives commonly found in supplements of this type. The doses typically used in these procedures can vary widely, but are usually in the range of about 2 mg per kg body weight to about 2 000 mg per kg body weight and frequently in the range of about 5 to about 500 mg per kg.

The food composition may preferably contain a vehicle for transporting the FSNMU through the large intestine or other part of the gastrointestinal tract. The vehicle may be a stable starch incorporated in an amount of 2 to 20% by weight. Incorporation may occur with the food ingredients during drying, for example by freeze-drying. Incorporation may also occur by microencapsulation. A typical process for microencapsulation requires first heating a low melting point oil above its melting point (< 40ºC), then adding the FSNMU to the mixture and also adding the resistant starch with the remaining ingredients and binders such as the gelatin and gums and atomising the dispersion in the top of a cooling tower to form uniform particles with a mean grain size typically in the range of 20 to 200 micrometres (µm). A preferred form of resistant starch is a high amylose starch, for example the compounds described in WO 94/03 049 and WO 94/14 342.

In the field of human and animal cosmetics, the invention also relates to the use of FSNMU for the preparation of a galenic form conventionally used for topical or oral application in hygiene, in particular in the form of oily, alcoholic or aqueous-alcoholic solutions, gels, water-in-oil emulsions or oil-in-water emulsions with the appearance of a cream or gel, optionally capable of foaming, in aerosol form or in the form of vesicle dispersions containing ionic and/or non-ionic lipids. These galenic forms are in all cases prepared according to the processes customary in the cosmetic field concerned. The composition may be, for example and in particular, a composition for cleansing, protecting, treating or caring for the scalp, a composition for the face, neck, hands or body (for example in the form of a vanishing cream type cream, night cream, make-up removing cream, sun protection cream or oil, body or face oil, cleansing milk, make-up removing milk or body milk), a make-up composition (for example a foundation), a composition for artificial tanning, a bath composition, a shampoo (treatment of the scalp) or a composition for oral hygiene (mouthwash, toothpaste or chewing gum).

In the field of human or veterinary pharmacy, and in particular in dermatology, otorhinolaryngology, ophthalmology, gastroenterology and gynaecology, the composition may be, for example, a capsule, a gelatin capsule, an emulsion, an ointment, a composition that can be injected under the skin, an ointment, a syrup, an atomiser, an eyewash, a shampoo or a mouthwash containing the FSNMU for the treatment or prophylaxis of the skin, eyes or mucous membranes. The various possible galenic forms are in all cases prepared according to the processes customary in the pharmaceutical field concerned.

The present invention is described in more detail by means of the following examples. It is to be understood that the examples are only intended to illustrate the invention and do not limit the invention in any way. Unless otherwise stated, the percentages are by weight.

Example 1 Metabolism of 20 : 3(5,11,14)

The determination of the profile of 5,11,14 in the cell line of immortalized human keratinocytes DK2-NR (described in the patent PCT/EP 96/05 812) is carried out in the following manner.

Confluent DK2-NR cultures are incubated for 4 days in NR-2 medium (Biofluids Inc., Rockville, MD, USA) with increased Ca²⁺ concentration (CaCl⁺ : 1.5 mM). The keratinocytes are then incubated twice for 2 days (exchange of medium with fatty acids after 2 days) in NR-2 containing 1 mg/ml bovine serum albumin (RSA, free fatty acid, Sigma Inc. St. Louis, USA) and 15 mM 5,11,14-eicosatrienoic acid methyl ester (5,11,14-ester) and/or arachidonic acid methyl ester (20:4n-6-ester).

Furthermore, the cells are pre-incubated for two days with 15 mM 20:4n-6-ester (see table, numbers 6 to 9) and then twice for 2 days with 5,11,14-ester and with a mixture of 20:4n-6-ester and 5,11,14-ester.

At the end of the incubation period, the cells are washed in HBSS (Hank's buffered saline) (Gibco, Life Technologies) containing 0.1% BSA and twice in HBSS. The cells are collected by scraping (cell scraper, Costar Inc.) the cells in the culture vessels. Then, the cells are centrifuged in 1 ml of HBSS (1,000 rpm). Cell extraction is carried out in a mixture of hexane and isopropanol (2:1 v/v) containing 2,6-di-t-butyl-p-cresol. The phospholipids are separated by chromatography (silica gel-60) with the eluent chloroform/acetone (96:4 v/v). The fatty acid fraction of the phospholipids is then esterified by heating (100ºC) in a solution containing 10% BF₃-methanol (Supelco, Bellefonte, PA, USA). The methyl esters are separated. The fatty acids are quantitatively determined using internal standards.

The results shown in Table 2 below show that 5,11,14-eicosatrienoic acid is a component of the total lipids (3.34%). Table 2

Example 2 Effect of 5,11,14-eicosatrienoic acid on the secretion of TNF-α by immortalized human keratinocytes treated with UV-B radiation

Immortalized human keratinocytes (DK2-NR) are incubated in culture vessels (diameter 3.5 cm) with 1.5 ml NR-2 medium. After reaching confluence, the Ca2+ concentration in the NR-2 medium is brought to 1.5 mM. After 4 days, the cells are incubated twice for 2 days in NR-2 without hydrocortisone with a high Ca2+ concentration (1.5 mM) and various fatty acids (namely 18:2n-6, 18:3n-3, 20:3 (5, 11,14)). At the end of the incubation period, the NR-2 medium is removed and stored at 37ºC to be used again as a culture medium for the cells after treatment with UV-B radiation (NR-2 conditioned medium). The cell cultures are washed twice with HBSS. For UV-B treatment, the cells are incubated in 0.5 ml of HBSS. The cell cultures are treated with UV-B radiation at 1.5 kJ (Phillips, TL100, maximum emission at 313 nm). The HBSS is then replaced with the corresponding NR-2 conditioned medium. After 24 hours, the supernatant is collected and stored at -20ºC. The concentration of secreted TNF-α in the supernatant is quantified by ELISA (Pelikine Compact, CLB Amsterdam, The Netherlands).

It can be found that, as indicated in Table 3, 5,11,14-eicosatrienoic acid inhibits the secretion of TNF-α (9 pg/ml) in the immortalized human keratinocytes treated with UV-B radiation compared to the control samples (11.6 pg/ml). Table 3

Example 3 Anti-inflammatory effect of additional FSNMU

The determination of the anti-inflammatory activity of 18:3(5,11,14), 18:3(3,9,12), 19:3(5,11,14), 19:3(4,10,13), 21:3(5,11,14), 21:3(6, 12,15), 22:3(5,11,14) and 22:3(7,13,16) was carried out on the cell line of immortalized human keratinocytes DK2-NR (described in the patent PCT/EP 96/05812) in the manner described above in Example 2. The results show the anti-inflammatory effects.

Example 4 Anti-inflammatory activity of Podocarpus nagi methyl ester following its topical application in mice.

The anti-inflammatory activity of Podocarpus nagi methyl ester (which has 26% C20:3 5,11,14 and contains 0.5% α-tocopherol and 0.2% ascorbyl palmitate) was determined in an arachidonic acid-induced edema test on the ear of a mouse.

Podocarpus nagi methyl ester is obtained from the oil extracted from the seeds of Podocarpus nagi, the extraction being carried out according to the procedure described in the two paragraphs following Table 1 of the description.

The following precise procedure is used:

Podocarpus nagi methyl ester or control oil of Podocarpus nagi is administered topically to the right ear of the mouse once daily for 5 days at a concentration of 20% (4 micrograms in 20 microliters of acetone). Each group consists of 5 mice; one group receives acetone alone. One hour after the last treatment, arachidonic acid is administered at a concentration of 2% in acetone. One hour after arachidonic acid administration, the thickness of the ear is determined using a micrometer (Oditest). The control oil of Podocarpus nagi is the same as Podocarpus nagi methyl ester described above, with C20:3 5,11,15 essentially replaced by oleic acid (C18:1n-9).

Results:

The edema of the ear corresponds to the increase in the thickness of the ear compared to the group that received only acetone.

SAM: Standard deviation of the mean

Indomethacin, a non-steroidal anti-inflammatory drug used as a positive control of the test at a concentration of 5% in acetone, strongly inhibits arachidonic acid-induced edema. Podocarpus nagi methyl ester at a concentration of 20% inhibits arachidonic acid-induced edema by 57%. The results show that Podocarpus nagi methyl ester has anti-inflammatory activity when administered topically to the skin of mice.

Example 5 Analysis of skin lipids after topical application of Podocarpus nagi ethyl ester in mice.

Podocarpus nagi ethyl ester is obtained from the oil extracted from the seeds of Podocarpus nagi, the extraction being carried out according to the procedure described in the two paragraphs following Table 1 of the description.

The following precise procedure is used:

The Podocarpus nagi ethyl ester (with antioxidants) or the control mixture (whose composition corresponds to the Podocarpus nagi ethyl ester, but C20:3 5,11,14 is essentially replaced by C18:1n-9) is administered topically to the right ear of the mouse once a day for 5 days at a concentration of 20% (4 micrograms in 20 microliters of acetone). Each group consists of 4 mice. Only acetone is applied to the left ear of the mice. 2 hours after the last application, the mice are euthanized. Tissue samples are taken from the treated ears for analysis of skin lipids.

The uptake of C20 : 3 5,11,14 into the phospholipids of the skin of the mouse ear was analyzed as follows:

Extraction of lipids: Extracts are obtained from 20 mg of each tissue sample using 0.8 ml H₂O, 1 ml CHCl₃ and 2 ml MeOH. After centrifugation (3 min at 1000 g), the residue is subjected to another extraction with 1 ml CHCl₃ and the supernatant is filtered over a glass frit and washed with 1 ml KCl (0.88%). The organic phase is extracted, concentrated under a stream of nitrogen and redissolved in 2 ml CHCl₃.

Separation of phospholipids (PL) and neutral lipids (NL): The different lipid classes were separated by solid phase chromatography on 3 ml columns containing 500 mg of silica (Supelco 57010). The samples described above (in 2 ml of CHCl₃) were added to the washed columns. The NL were recovered with 2 ml of CHCl₃, the PL with 4 ml of CHCl₃/MeOH (2/1) and 4 ml of MeOH. The samples are concentrated under a stream of nitrogen and redissolved in 10 ml of CHCl₃.

Thin layer chromatography of phospholipids: The PL samples obtained above are placed on 10 x 10 thin layer chromatography plates (Merck 1.13727) and the PL are separated in CHCl3/MeOH/acetic acid/H2O (50/37.5/3.5/2.9). The PL are identified with reference samples.

Methylation of phospholipids: The samples are then methylated with 200 μl methanolic HCl/hexane (4/1) containing 0.4 μg C17:0 for 16 hours at 65°C. They are then neutralized with 0.5 ml K₂CO₃ (6%). For analysis by gas chromatography, the methyl esters (ME) are extracted twice with 300 μl hexane, concentrated under a stream of nitrogen and redissolved in 5 μl isooctane.

Gas chromatography of phospholipids: MEs are injected into a Hewlett Packard GC model 6890 equipped with a sample collector and a capillary column. RESULTS: Fatty acids in the lipid pools after topical application of the control ethyl esters* (control) and Podocarpus nagi ethyl esters (seeds) to the mouse ear.

* Saffron/Sunflower/Apricot (43/7/50)

** Antioxidants: 0.5% w/w α-tocopherol and 0.2% w/w ascorbyl palmitate

C20 : 3 5,11,14 increases in the phospholipids, while 20 : 4n-6 (arachidonic acid) decreases. The results show that C20 : 3 5,11,14 is incorporated into the skin phospholipids in mice after topical application.

The replacement of 20 : 4n-6 with C20 : 3 5,11,14 appears to correlate with the anti-inflammatory properties of C20 : 3 5,11,14, which were demonstrated after topical application in the experiment in which edema is induced in the ear of a mouse by arachidonic acid (Example 4).

Example 6 Body milk (oil-in-water emulsion) Oil phase:

Glyceryl stearate/ PEG-100 stearate (Arlacel 165, sold by ICI) (emulsifier) 1%

Polysorbate 60 (emulsifier) 0.8%

Hydrogenated polyisobutene 2%

Stearic acid 1%

Ephedra campylopoda seed oil 8%.

Aqueous phase:

Glycerine 3%

Carbomer (Carbopol 941, available from Goodrich) (thickener) 0.3%

Triethanolamine (neutralizing agent) 0.3%

Preservative 0.3%

Water ad 100%.

The emulsion is made by incorporating the oil phase into the water phase while stirring constantly. The body milk helps to provide good protection for the skin against inflammation.

Example 7 Care fluid (oil-in-water emulsion) Oil phase:

Sesquistearate of methyl glucose (emulsifier) 2%

Cyclomethicone 13%

Podocarpus nagi seed oil 2%

Perfume 0.2%

Sesquistearate of methylglucose-PEG 20 (emulsifier) 2%.

Aqueous phase:

Xanthan gum (thickener) 0.2%

Polyacrylamide/C₁₃₋₁₄-Isopraffin/Laureth-7 (Sepigel 305, sold by Seppic) (thickener) 0.8%

Preservative 0.3%

Water ad 100%.

The emulsion is prepared according to the procedure described in Example 6. A white fluid is obtained which helps to provide good protection of the skin against inflammation.

Example 8 Care cream (water-in-oil emulsion) Oil phase: (A)

Polyglyceryl-4 isostearate/cetyl dimethicone copolyol/hexyl laurate (Abil WE 09, sold by Goldschmidt) (emulsifier) 4%

Isohexadecane 5%

Caltha palustris seed oil 10%

Cyclomethicone 3.5%

5-(n-octanoyl)-salicylic acid 1%

Perfume 0.15%.

Aqueous phase: (B)

Glycerine 10%

Cellulose gum 0.5%

Magnesium sulfate 0.65%

Preservative 0.3%

Water ad 100%.

To prepare the emulsion, the components of phase A are heated to 80ºC until they are completely dissolved, after which they are cooled to 65ºC. Then phase B is heated to 65ºC, phase A is poured into phase B while stirring constantly and the mixture is cooled. A white fluid is obtained that helps to protect the skin well against inflammation.

Claims (3)

1. Use of at least one FSNMU of the following formula, wherein the FSNMU is in the form of an acid, a salt or an ester, R¹ represents a C₁₋₅ alkyl group and R² represents a C₂₋₆ alkyl group: for the manufacture of a composition intended to modulate lipid metabolism in the superficial tissues of mammals. 2. Use according to claim 1 for the preparation of a composition intended for the treatment or prevention of inflammation in the superficial tissues of mammals. 3. Use according to claim 1 or 2, wherein the FSNMU is 20:3(5,11,14).